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首页> 美国卫生研究院文献>The Journal of Biological Chemistry >Transmembrane Segment II of NhaA Na+/H+ Antiporter Lines the Cation Passage and Asp65 Is Critical for pH Activation of the Antiporter
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Transmembrane Segment II of NhaA Na+/H+ Antiporter Lines the Cation Passage and Asp65 Is Critical for pH Activation of the Antiporter

机译：NhaA Na + / H +反转运蛋白的跨膜片段II排列阳离子通道而Asp65对于反转运蛋白的pH激活至关重要

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摘要

The crystal structure of Escherichia coli NhaA determined at pH 4 has provided insights into the mechanism of activity of a pH-regulated Na+/H+ antiporter. However, because NhaA is activated at physiological pH (pH 5.5–8.5), many questions related to the active state of NhaA have remained elusive. Our experimental results at physiological pH and computational analyses reveal that amino acid residues in transmembrane segment II contribute to the cation pathway of NhaA and its pH regulation: 1) transmembrane segment II is a highly conserved helix and the conserved amino acid residues are located on one side of the helix facing either the cytoplasmic or periplasmic funnels of NhaA structure. 2) Cys replacements of the conserved residues and measuring their antiporter activity in everted membrane vesicles showed that D65C, L67C, E78C, and E82C increased the apparent Km to Na+ and Li+ and changed the pH response of the antiporter. 3) Introduced Cys replacements, L60C, N64C, F71C, F72C, and E78C, were significantly alkylated by [14C]N-ethylmaleimide implying the presence of water-filled cavities in NhaA. 4) Several Cys replacements were modified by MTSES and/or MTSET, membrane impermeant, negatively and positively charged reagents, respectively, that could reach Cys replacements from the periplasm only via water-filled funnel(s). Remarkably, the reactivity of D65C to MTSES increased with increasing pH and chemical modification by MTSES but not by MTSET, decreased the apparent Km of the antiporter at pH 7.5 (10-fold) but not at pH 8.5, implying the importance of Asp65 negative charge for pH activation of the antiporter.
机译：在pH 4下测定的大肠杆菌NhaA的晶体结构为了解pH调节的Na + / H + 反向转运蛋白的活性机理提供了见识。但是，由于NhaA在生理pH值（pH 5.5-8.5）下被激活，因此许多与NhaA活性状态有关的问题仍然难以捉摸。我们在生理pH值上的实验结果和计算分析表明，跨膜片段II中的氨基酸残基有助于NhaA的阳离子途径及其pH调节：1）跨膜片段II是高度保守的螺旋，保守的氨基酸残基位于一个螺旋的一侧面向NhaA结构的细胞质或周质漏斗。 2）Cys置换保守残基并测量其在外翻膜囊泡中的抗转运蛋白活性表明，D65C，L67C，E78C和E82C使K Kup>增加到Na + 和Li + 并改变了反向转运蛋白的pH响应。 3）引入的Cys替代品L60C，N64C，F71C，F72C和E78C被[ 14 C] N-乙基马来酰亚胺显着烷基化，这表明NhaA中存在充满水的空腔。 4）分别用MTSES和/或MTSET，膜不渗透剂，带负电荷和带正电荷的试剂修饰了几种Cys替代物，这些替代物只能通过充水漏斗从周质达到Cys替代物。值得注意的是，D65C对MTSES的反应性随pH值的增加而增加，并且通过MTSES而不是MTSET的化学修饰而增加，在pH 7.5（10倍）但在pH 8.5时，反转运蛋白的表观Km降低，这表明Asp 65 负电荷可激活反向转运蛋白的pH。

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